How does your transformation efficiency compare with the different teams? Calcul

Question

How does your transformation efficiency compare with the different teams? Calculate the transformation efficiency of the following experiment using the information and results listed below: DNA plasmid concentration = 0.08ug/ul 250ul CaC12 transformation solution 10ul pGLO plasmid solution 250ul LB Broth 100ul cells spread on agar 227 colonies of transformants Show your calculations, and answer the following: What number of colonies were on the LB/amp/are plate? How much DNA was spread on the plate in ug? What is the transformation efficiency? If a particular experiment known to have 3 times 10 bacteria/ug DNA, how many transformant colonies would be expected to grow on the LB/amp/are plate? You can assume that the concentration of DNA and fraction of cells spread on the LB agar are the same as that of the pGLO laboratory.

Explanation / Answer

question 1 --Different tranformation protocol are used in laboratories for checking the efficiency and concentration of plasmid used in molecular biology.The range of number of colonies after transformation are from 800-7000. The different methods include calcium chloride method, inoue method of transformation.

question-2--DNA plasmid concentration=0.08microgram/microlitre, total plasmid DNA concentration=0.08 x 10=0.8 microgram of pGLO in 10 microlitre of solution.

volume of solution sread on agar plate=100 microlitre, so total volume=100+10 =110 microlitre.

250 microlitre of calcium chloride transformation solution in 250 microlitre of LB media and 10 microlitre of plasmid pGLO solution, so total volume=510microlitre.

so ratio of bacterial cells spread on plate/ total volume used for transformation with pGLO plasmid=100/510=0.196.

plasmid pGLO concentration x total volume of transformation=0.8 x 0.196=0.1568 microgram.

Answer--transformation efficiency=volume of bacterial cells plated/plasmid pGLO concentration in a defined volume used for transformation=100/0.1568=637.75=6.37x 102 colonies/microgram of pGLO plasmid.

1)answer--number of colonies on LB plate/amp/arabinose=6.37 x 102 colonies per microgram of pGLO plasmid used for transformation.

2)answer--plasmid DNA spread on the LB plate=0.1568 microgram.

3)answer--transfromation efficiency=6.37 x 102colonies per microgram of pGLO plasmid used for transfromation.

question-3--number of colonies of bacteria--3 x 10 bacteria/microgram of DNA=30 bacteria/microgram of plasmid DNA, volume of plasmid DNA used for transformation=10 microlitre, volume spread on LB agar plate=100 microlitre, concentration of plasmid DNA in 10 microlitre volume solution=0.8 microgram.

1 microgram of plasmid DNA present in 30 bacteria colonies, so 0.8 microgram in 10microlitre of plasmid DNA solution contain 30 x 0.8=24 bacteria colonies in 100 microlitre of solution spread on LB plate.

answer-24 transformation bacteria colonies in 100 microlitre of solution spread on plate.

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